FLAVONOID DERIVATIVE COMPOUNDS AND METHOD FOR PREPARING SAME BY DEPOLYMERIZATION OF CONDENSED TANNINS

Abstract

A compound of general formula (I) in which R.sub.1, R.sub.2, R.sub.3 and R.sub.5, identical or different, each represent a hydrogen atom or a hydroxyl group, optionally protected by a protecting group. R.sub.4 represents a hydrogen atom or an —OR.sub.7 group, in which R.sub.7 represents a hydrogen atom, a protecting group or a gallate group. R.sub.6 represents a hydroxyl group, optionally protected by a protecting group. R′.sub.1, R′.sub.2, R′.sub.3 and R′.sub.4, identical or different, each represent a hydrogen atom or a substituent not comprising a mesomeric electron withdrawing group conjugated with the furan ring. A substituent from R′.sub.1, R′.sub.2, R′.sub.3 and R′.sub.4 representing the covalent bond with the pyran ring or one of the salts of same. A method for obtaining such a compound includes a step of depolymerization of condensed tannins in the presence of an acid by means of a nucleophile derived from furan.

##STR00001##

Claims

1. A compound of general formula (I): ##STR00026## in which: R.sub.1, R.sub.2, R.sub.3 and R.sub.5, which may be identical or different, each represent a hydrogen atom or a hydroxyl group, optionally protected by a hydroxyl-function-protecting group, R.sub.4 represents a hydrogen atom or an —OR.sub.7 group, in which R.sub.7 represents a hydrogen atom, a hydroxyl-function-protecting group, or a group of general formula (II): ##STR00027## in which R″.sub.1, R″.sub.2 and R″.sub.3, which may be identical or different, each represent a hydrogen atom or a hydroxyl-function-protecting group, R.sub.6 represents a hydroxyl group, optionnally protected by a hydroxyl-function-protecting group, and R′.sub.1, R′.sub.2, R′.sub.3 and R′.sub.4, which may be identical or different, each represent a hydrogen atom or a substituent not comprising any mesomeric-effect electron-withdrawing group conjugated to the furan nucleus, a substituent among R′.sub.1, R′.sub.2, R′.sub.3 and R′.sub.4 representing the covalent bond with the pyran ring, or a salt thereof

2. The compound as claimed in claim 1, in which R′.sub.1, R′.sub.2, R′.sub.3 and R′.sub.4, which may be identical or different, each represent, with the exception of the substituent forming the covalent bond with the pyran ring: a hydrogen atom, a group comprising an electron-donating radical bonded directly or by conjugation to the furan ring, which is optionally substituted, or a linear, branched or cyclic carbon-based radical, comprising a single ring or several condensed rings, which is saturated or unsaturated, optionally aromatic, which is optionally substituted, optionally comprising one or more heteroatoms or one or more groups comprising one or more heteroatoms.

3. The compound as claimed in claim 1, in which at least one substituent among R′.sub.1, R′.sub.2, R′.sub.3 and R′.sub.4 represents a hydrogen atom.

4. The compound as claimed in claim 1, in which R′.sub.1 represents the covalent bond with the pyran ring.

5. The compound as claimed in claim 1, in which R′.sub.3 and R′.sub.4 each represent a hydrogen atom.

6. The compound as claimed in claim 1, in which R′.sub.2 represents a hydrogen atom or an alkyl group, which is preferably C.sub.1-C.sub.18, and preferentially C .sub.1-C.sub.4.

7. A method for obtaining a compound as claimed in claim 1, comprising a depolymerization step of depolymerizing condensed tannins in the presence of an acid by means of a nucleophile of general formula (III): ##STR00028## in which R′.sub.1, R′.sub.2, R′.sub.3 and R′.sub.4, which may be identical or different, each represent a hydrogen atom or a substituent not comprising any mesomeric-effect electron-withdrawing group conjugated to the furan nucleus, at least one substituent among R′.sub.1, R′.sub.2, R′.sub.3 and R′.sub.4 representing a hydrogen atom.

8. The method as claimed in claim 7, wherein, for the depolymerization step, a nucleophile of general formula (III)/depolymerizable condensed tannins molar ratio is greater than or equal to a nucleophile of general formula (III)/dimer B2 molar ratio required to obtain at least 2.5% of compounds of general formula (I), relative to a total of the compounds formed during a reaction of depolymerization of a dimer B2 by the nucleophile of general formula (III) in a presence of hydrochloric acid at 0.1 N in methanol, at 30° C. and in a reaction time of 10 minutes.

9. The method as claimed in claim 7, wherein R′.sub.1, R′.sub.2, R′.sub.3 and R′.sub.4, which may be identical or different, each represent: a hydrogen atom, a group comprising an electron-donating radical bonded directly or by conjugation to the furan ring, which is optionally substituted, or a linear, branched or cyclic carbon-based radical, comprising a single ring or several condensed rings, which is saturated or unsaturated, optionally aromatic, which is optionally substituted, optionally comprising one or more heteroatoms or one or more groups comprising one or more heteroatoms.

10. The method as claimed in claim 7, wherein, in general formula (III), at least R′.sub.1 represents a hydrogen atom.

11. The method as claimed in claim 7, wherein, in general formula (III), at least R′.sub.3 and R′.sub.4 each represent a hydrogen atom.

12. The method as claimed in claim 7, in which R′.sub.2 represents a hydrogen atom or an alkyl group, which is preferably C.sub.1-C.sub.18, preferentially C.sub.1-C.sub.4.

13. The method as claimed in claim 7, wherein, for the depolymerization step, the acid is used in a concentration equivalent to its concentration required to confer a pH of between −1 and 3.5 on an aqueous solution.

14. The method as claimed in claim 7, wherein the depolymerization step is carried out in a polar, solvent or in a mixture of solvents containing at least one polar, solvent.

15. The method as claimed in claim 7, wherein the depolymerization step is carried out at a temperature below or equal to a boiling point of the nucleophile of general formula (III) at an applied pressure, and at less than or equal to a boiling point of the solvent at said applied pressure.

16. The method as claimed in claim 7, comprising a prior step of extracting the condensed tannins from biomass.

17. The method as claimed in claim 7, wherein the depolymerization step is carried out directly from biomass.

18. A method for depolymerizing condensed tannins using a compound of general formula (III): ##STR00029## in which R′.sub.1, R′.sub.2, R′.sub.3 and R′.sub.4, which may be identical or different, each represent a hydrogen atom or a substituent not comprising any mesomeric-effect electron-withdrawing group conjugated to the furan nucleus, at least one substituent among R′.sub.1, R′.sub.2, R′.sub.3 and R′.sub.4 representing a hydrogen atom.

19. The method as claimed in claim 18, wherein, in general formula (III), at least R′.sub.1 represents a hydrogen atom.

20. The method as claimed in claim 18, wherein, in general formula (III), at least R′.sub.3 and R′.sub.4 each represent a hydrogen atom.

21. The method as claimed in claim 18, wherein R′.sub.2 represents a hydrogen atom or an alkyl group, which is preferably C.sub.1-C.sub.18, and preferentially C.sub.1-C.sub.4.

Description

[0137] The features and advantages of the invention will emerge more clearly in the light of the examples below, which are given simply by way of illustration and are in no way limiting of the invention, with the support of FIGS. 1 to 8, in which:

[0138] FIG. 1 shows a UPLC chromatogram at 280 nm of the reaction crude obtained after carrying out a condensed tannin depolymerization method according to one particular embodiment of the present invention (furanolysis), using hydrochloric acid (HCl) as acid;

[0139] FIG. 2 represents a graph showing the amounts of each of the reaction products, per gram of tannins treated, for condensed tannin depolymerization methods according to particular embodiments of the present invention (furanolysis), using hydrochloric acid (HCl) or methanesulfonic acid (MsOH) as acid, and also for a tannin depolymerization reaction using a sulfur-bearing nucleophile proposed by the prior art (thiolysis);

[0140] FIG. 3 represents a graph showing the effect of the initial concentration of dimer B2 on its kinetics of disappearance during a method, in accordance with one embodiment of the invention, of furanolysis of a commercial preparation of dimer B2 in the presence of a methanolic solution of furan (25% v/v) and of HCl (0.1 mol.l.sup.−1);

[0141] FIG. 4 represents a graph showing the effect of the initial concentration of dimer B2 on the kinetics of appearance of the depolymerization products during the furanolysis method having been carried out for obtaining FIG. 3;

[0142] FIG. 5 shows a UPLC chromatogram at 280 nm of the reaction crude obtained after carrying out a condensed tannin depolymerization method according to one particular embodiment of the present invention (sylvanolysis), using hydrochloric acid (HCl) as acid;

[0143] FIG. 6 shows UPLC chromatograms, with detection at 280 nm, carried out on an aqueous solution of the products resulting from the depolymerization of a condensed tannin extract by thiolysis, (a) before, and (b) after, bringing into contact for 19 h with a buffer solution at pH 9;

[0144] FIG. 7 shows UPLC chromatograms, with detection at 280 nm, carried out on an aqueous solution of a crude product resulting from the depolymerization of a condensed tannin extract using sylvan in accordance with one particular embodiment of the invention, (a) before, and (b) after, bringing into contact for 19 h with a buffer solution at pH 9; and

[0145] FIG. 8 shows sigmoid curves established from the various experimental results, expressing the molar ratio between the derivatives derived from the extension units U.sub.ext and the total of the extension units and of the terminal units U.sub.term (U.sub.ext/(U.sub.ext+U.sub.term)), as a function of the “nucleophile/dimer B2” initial molar ratio, for experiments in which the dimer B2 was depolymerized using the nucleophiles in accordance with the invention, furan and sylvan, for initial concentrations of dimer B2 of 100, 200 or 400 mg.l.sup.−1, and various initial nucleophile concentrations.

MATERIALS AND METHODS

[0146] For the experiments below, the solvents and reagents were obtained from Sigma-Aldrich for the furan, the sylvan, the 2-mercaptoethanol, the anhydrous methanesulfonic acid (MsOH) and the catechin; from Carl Roth for the fuming hydrochloric acid (HCl 37% aq.); and from ProLabo for the methanol (analytical grade) (MeOH) and the absolute ethanol (EtOH). The “hydrochloric” and “methanesulfonic” solutions are prepared respectively by a dilution of fuming HCl (37% aq.) and of MsOH in the solvent concerned.

[0147] The industrial seed condensed tannin extracts produced from marcs originating from vinifications into white wines (“white seed tannins”) (high quality grade) were obtained from the Union des Distilleries de la Méditerranée [Union of Mediterranean Distilleries], and the dimer B2 from the company Extrasynthése.

General Protocol for Analyzing the Depolymerization Reaction Products (Identification and Quantification)

[0148] The analysis consists in carrying out a separation of the depolymerization reaction products by ultra performance liquid chromatography (Waters UPLC system) coupled in series to a diode array detector (DAD) and to a mass spectrometer (Brucker AmaZonX model) (UPLC-MS). The samples resulting from the depolymerization are analyzed extemporaneously, without pretreatment, the reaction medium being diluted, where appropriate, for a final tannin concentration at 1 g.l.sup.−1 (200 mg.l.sup.−1 for the dimer B2). The samples (2 μl) are injected on to a Waters Acquity Atlantis HSS T3 1.8 μm−2.1×100 mm column, and eluted with the solvents A (H.sub.2O:HCOOH 99:1) and B (H.sub.2O:HCOOH:MeCN 19:1:80), according to the A/B gradient: 99.9% to 60% linear, 5 min; 60% to 1% linear, 2 min; 1% isocratic, 1 min; 1% to 99.9% linear, 1 min. The UV chromatogram recorded at 280 nm allows the quantitative analysis by integration of the peaks corresponding to each of the products, by virtue of a prior external calibration of the reaction products. The MS(+) chromatogram allows the identification of the products, on the basis of the m/z values.

[0149] A preparative separation by flash chromatography on a “DIOL” grafted silica column (Interchim), followed where appropriate by a semi-preparative chromatography (C18 grafted silica), makes it possible to isolate the products with a high purity in order to confirm the structures by NMR and to determine their coefficients of response on the UPLC-MS apparatus.

[0150] For each kinetics undertaken, the quantification of the reagents and of the products is carried out by UPLC-MS(+) analysis. To this effect, as soon as the reagents are brought together, the reaction mixture is immediately distributed by sampling one and the same volume in several tubes. All the samples are placed in a thermostated bath and brought to the selected temperature. One sample is sacrificed for each point of the time course corresponding to a given time. Once removed from the bath, the sample is cooled to 10° C., diluted if necessary and injected extemporaneously into the chromatograph.

EXAMPLE 1

Comparative Study of Processes for Thiolysis or for Furanolysis of White Seed Tannins at 1 g/l

[0151] The polymerization of an extract of white seed tannins was carried out in an acidic medium by means, on the one hand, of a sulfur-bearing nucleophile as proposed by the prior art (analytical thiolysis reaction, comparative example) and, on the other hand, of a nucleophile in accordance with the present invention, furan (furanolysis reaction), in the presence respectively of two different acids, hydrochloric acid and methanesulfonic acid.

Thiolysis (Comparative Example)

[0152] An optimized thiolysis reaction, according to the protocol described in the publication by Roumeas et al., 2013, is carried out in the following way.

[0153] In a 10 ml tube, the tannin extract (10 mg) is dissolved in methanol (MeOH) (5 ml), then 5 ml of the thiolytic depolymerization solution (12 μl of 2-mercaptoethanol and 83 μl of fuming HCl per 5 ml of MeOH qs) are added. The tube is closed with a phenolic stopper equipped with a PTFE septum, and left to stir at 40° C. for 2 h.

Furanolysis—HCl

[0154] A first furanolysis reaction in accordance with the present invention is carried out as follows.

[0155] In a 10 ml tube, the tannin extract (10 mg) is dissolved in MeOH (5 ml), then the furan (2.5 ml) and a hydrochloric solution of MeOH (0.4 mol.l.sup.−1) are added. The tube is closed with a phenolic stopper equipped with a PTFE septum, and left to stir at 40° C. for 1 h.

Furanolysis—MsOH

[0156] A second furanolysis reaction in accordance with the present invention is carried out as follows.

[0157] In a 10 ml tube, the tannin extract (10 mg) is dissolved in MeOH (5 ml), then furan (2.5 ml) and the solution of MsOH at 0.4 mol.l.sup.−1 in MeOH (5 ml) are added. The tube is closed with a phenolic stopper equipped with a PTFE septum, and left to stir at 40° C. for 1 h.

[0158] In the grape, since a proportion of the constituent units of the polymer chains forming the tannins are galloylated, that is to say esterified with gallic acid at the level of the hydroxyl radical in the C3 position, the depolymerization reactions result in the formation of the following products: [0159] furylated extension units corresponding to general formula (I) according to the invention:

##STR00022## [0160] terminal units:

##STR00023##

[0161] wherein Gal represents a gallate group of formula (II) defined above.

[0162] For each of the depolymerization reactions, the amount of each of the products formed is determined by UPLC-MS, as indicated above. The depolymerization products are referenced as follows: catechin terminal units (Cat), epicatechin terminal units (Ec), epicatechin gallate terminal units (EcG), and furylated extension units of catechin (Cat-F), of epicatechin (Ec-F) and of epicatechin gallate (EcG-F).

[0163] By way of example, FIG. 1 shows the UPLC chromatogram at 280 nm of the reaction crude obtained at the end of the furanolysis—HCl reaction described above.

[0164] For each of the products formed, the retention time and the m/z value observed by MS(+) are indicated in table 1 below.

TABLE-US-00001 TABLE 1 UPLC retention time and m/z values observed by MS(+) of the depolymerization products obtained by means of a furanolysis - HCl method in accordance with the invention Product Retention time (min) (M + H.sup.+) Cat 2.8 291 Ec 3.2 291 EcG 4.0 443 Cat-F 3.9 357 Ec-F 4.7 357 EcG-F 5.3 509

[0165] The amounts of each of the reaction products formed per gram of tannins are deduced from this analysis, for each of the processes described above. The results obtained are shown in FIG. 2.

[0166] It is observed that the reactions carried out with furan as nucleophile, in accordance with the present invention, give results similar to the depolymerization reactions of the prior art by optimized thiolysis, and with a yield that is even better with regard to the terminal units.

[0167] Moreover, no significant difference is observed between the use of HCl or of MsOH as acid catalyst.

EXAMPLE 2

Depolymerization by Furanolysis of Douglas Pine Tannins

[0168] The pine bark lipids (5 g) are extracted with 3×50 ml of hexane. The tannins are then extracted with a 70/30/0.05 v/v/v acetone/H.sub.2O/TFA mixture (3×50 ml). The acetone is evaporated off under vacuum, and the aqueous phase is then lyophilized to give 900 mg of tannin extract in the form of a brownish powder.

[0169] A method for depolymerization of the tannins thus obtained in accordance with the invention is carried out as follows.

[0170] In a tube, the tannins (100 mg) are dissolved in MeOH (72.5 ml), and then furan (25 ml) and a hydrochloric methanol solution at 4.0 mol.l.sup.−1 (2.5 ml) are added. The tube is closed with a phenolic stopper equipped with a PTFE septum, and left to stir at 40° C. for 1 h.

[0171] The yields evaluated on the basis of a UPLC-MS analysis, as indicated above, are greater than or equal to those obtained by means of an analytical thiolysis (that is to say 440 μmol of furylated derivatives per gram of extract).

EXAMPLE 3

Furanolysis of the Dimer B2

[0172] The dimer B2 was subjected to a depolymerization step in accordance with the present invention, at several concentrations, according to the following reaction conditions.

[0173] For each test, the kinetics of the depolymerization reaction is monitored over time by determination by UPLC-MS, as described above, of the concentrations of unreacted dimer B2 and of reaction products formed.

[0174] 10 g.l.sup.−1

[0175] 15 mg of dimer B2 are dissolved in MeOH (1.12 ml), then furan (375 μl) and then fuming HCl (12.5 μl) are added. The solution (180 μl) is transferred into 300 μl vials, which are sealed with aluminum capsules with a septum, and brought to 40° C. The reaction is monitored by UPLC-MS(+) for 8 h.

[0176] 1 g.l.sup.−1

[0177] 1.5 ml of solution of dimer B2 at 1.0 g.l.sup.−1 are prepared in a 25% v/v furan/MeOH mixture. The hydrochloric acid (12.5 μl) is added. 180 μl of solution are transferred into 300 μl vials, which are sealed with aluminum capsules with a septum, and brought to 40° C. The reaction is monitored by UPLC-MS(+) for 8 h.

[0178] 0.2 g.l.sup.−1

[0179] 1.5 ml of solution of dimer B2 at 200 mg.l.sup.−1 are prepared in a 25% v/v furan/MeOH mixture. The hydrochloric acid (12.5 μl) is added. 180 μl of solution are transferred into 300 μl vials, which are sealed with aluminum capsules with a septum, and brought to 40° C. The reaction is monitored by UPLC-MS(+) for 180 min.

[0180] For each initial concentration of dimer B2, the results obtained are shown in FIG. 3, for the dimer B2 disappearance kinetics, and in FIG. 4, for the depolymerization product appearance kinetics.

[0181] It is noted that the maximum amount of depolymerized units represents 90% and 97% of the initial amount of dimer B2, respectively for initial concentrations of 0.2 g/l and 1.0 g/l. These maximum amounts are obtained in very short reaction times, respectively of 10 min and 20 min. In comparison, lower yields are obtained when the initial concentration of dimer B2 is equal to 10 g/l, since the maximum amount of depolymerized units then represents 55% of the initial amount of dimer B2. In addition, the lower the initial concentration of dimer B2, the faster the appearance of the depolymerization products.

[0182] For a dimer B2 concentration of 10 g/l, the apparent kinetics of disappearance of the dimer B2 is in addition slower than the apparent rate of appearance of the depolymerized units. The difference in kinetics observed between the appearance of the depolymerization products and the disappearance of the dimer B2 suggests that the depolymerization products and the dimer B2 become competitive nucleophiles with respect to the furan at a high concentration of flavan units in the medium, that is to say at high concentrations of the dimer itself and its depolymerization products. These observations appear to indicate a reaction scheme involving numerous elementary reactions, and demonstrate the advantage of proceeding, when the nucleophile is the furan, and as recommended by the present invention, under dilute conditions, that is to say at low initial concentrations of condensed tannins in the reaction medium, of less than 10 g/l.

EXAMPLE 4

Furanolysis of White Seed Tannins Using the Furan in Ethanol

[0183] A method for depolymerizing white seed tannins is carried out in accordance with the present invention, by means of furan as nucleophile, in ethanol as solvent, according to the following operating protocol.

[0184] A solution of tannin sample at 2 g.l.sup.−1 in absolute ethanol (EtOH), and the furanolysis solution (50% v/v furan in methanesulfonic EtOH at 0.2 mol.l.sup.−1) are prepared. The reaction mixture is prepared by adding 1.0 ml of each solution to an Eppendorf tube, then 200 μl of solution are transferred into 300 μl vials, which are sealed with aluminum capsules with a septum, and brought to 40° C. The reaction is monitored by UPLC-MS(+) for 80 min. The maximum yield, obtained after 40 min of reaction, is 510 μmol of depolymerization products per gram of extract (that is to say 73% of the estimated maximum value by thiolysis).

EXAMPLE 5

Furanolysis of White Seed Tannins with Furan—Influence of the Furan/Tannin Ratio

[0185] Methods for depolymerizing white seed tannins are carried out in accordance with the present invention, by means of furan as nucleophile, with several different “moles of furan/weight of tannins” ratios, according to the following operating protocol.

[0186] The white seed tannin extract is dissolved in MeOH (12 ml). The furan (4 ml) and then the fuming hydrochloric acid (133 μI) are added. This solution is distributed into 8 Eppendorf tubes, in a proportion of 1.5 ml per tube. Each tube is closed with Parafilm® and brought to 30° C. The reaction kinetics are monitored by UPLC-MS(+) for 8 h.

[0187] The operating conditions and results obtained are shown in table 2 below.

TABLE-US-00002 TABLE 2 Operating conditions and yield of a reaction of furanolysis of white seed tannins with furan in accordance with the invention “Moles of furan/moles Max. yield Tannin “Moles of of tannin (compared concen- furan/g of eq. Max. yield with Time for tration tannins” monomers” (μmol .Math. g.sup.−1 analytical obtaining (g/l) ratio ratio of tannins) thiolysis) max. yield 1 3.44 1000 702 100% 1 h 30- 6 h 2 1.72 500 652 93% 1 h 30- 8 h 3 1.15 334 611 87% 1 h 30- 8 h 5 0.688 200 610 87% 4 h 30 10 0.344 100 461 66% 4-8 h

[0188] These tests show that, when the nucleophile is furan, high reaction yields can be obtained for all the tannin concentrations tested. These yields are highest for tannin concentrations of less than or equal to 5 g.l.sup.−1.

EXAMPLE 6

Depolymerization of White Seed Tannins with Sylvan—Influence of the Sylvan/Tannin Ratio

[0189] Methods for depolymerizing white seed tannins are carried out in accordance with the present invention, by means of sylvan as nucleophile, with several different “moles of sylvan/weight of tannins” ratios, according to the following operating protocol.

[0190] The white seed tannin extract is dissolved in MeOH (12 ml). The sylvan (4 ml) and then fuming hydrochloric acid (133 μl) are added. This solution is distributed into 8 Eppendorf tubes, in a proportion of 1.5 ml per tube. Each tube is closed with Parafilm® and brought to 30° C. The reaction kinetics are monitored by UPLC-MS(+) for 4 h.

[0191] The depolymerization products are referenced as follows: catechin terminal units (Cat), epicatechin terminal units (Ec), epicatechin gallate terminal units (EcG), and sylvanylated extension units of catechin (Cat-S), of epicatechin (Ec-S) and of epicatechin gallate (EcG-S).

[0192] FIG. 5 shows the UPLC chromatogram obtained after 1 h of reaction.

[0193] For each of the products formed, the retention time and the m/z value observed by MS(+) are indicated in table 3 below.

TABLE-US-00003 TABLE 3 UPLC retention time and m/z values observed by MS(+) of the depolymerization products obtained by means of a sylvanolysis - HCl method in accordance with the invention Product Retention time (min) (M + H.sup.+) Cat 2.8 291 Ec 3.2 291 EcG 4.0 443 Cat-S 4.5 371 Ec-S 5.4 371 EcG-S 5.9 523

[0194] The operating conditions and results obtained, in terms of yield, are indicated in table 4 below.

TABLE-US-00004 TABLE 4 Operating conditions and yield for a reaction of depolymerization of white seed tannins with sylvan in accordance with the invention “Moles of “Moles of sylvan/moles Max. yield Tannin sylvan/g of tannin (compared concen- of eq. Max. yield. with Time for tration tannins” monomers” (μmol .Math. g.sup.−1 analytical obtaining (g/l) ratio ratio of tannins) thiolysis) max. yield 1 2.77 800 720 103% 30 min- 1 h 30 5 0.555 160 730 104% 30 min- 2 h 10 0.277 80 650 93% 60 min 20 0.139 40 662 95% 45 min- 1 h 30 40 0.069 20 626 89% 45 min- 1 h 30 80 0.035 10 581 83% 90 min 1 0.0035 1 329 47% 4-6 h

[0195] These tests show that, when the nucleophile is sylvan, very high reaction yields can be obtained for all the tannin concentrations tested.

EXAMPLE 7

Preparative Depolymerization of White Seed Tannins with Furan

[0196] A depolymerization method in accordance with the invention is carried out in the following way.

[0197] In a bottle, the tannin extract (5.0 g) is dissolved in MeOH (200 ml), then the furan (108 ml), and then the hydrochloric methanol (83 ml of fuming HCl in 108 ml of MeOH) are added without stirring. The mixture is brought to 40° C., for 30 min, and then cooled to 0° C. 500 ml of an aqueous solution of Na.sub.2CO.sub.3 (106 g.l.sup.−1) are then added. The mixture is extracted with ethyl acetate (AcOEt) (3×400 ml), then subjected to evaporation. A pasty brown-black solid is obtained (3.34 g), which is taken up in diethyl ether (Et.sub.2O) (200 ml), triturated and sonicated, and then washed with brine (300 ml). These operations are repeated twice, and the solutions obtained are dried (Na.sub.2SO.sub.4) and then evaporated so as to obtain a brownish pasty solid (2.40 g), which consists of a mixture of terminal units and of furylated extension units.

[0198] The product thus obtained is purified by flash chromatography. The mixture (150 mg) is dissolved in Et.sub.2O (5 ml), and is then injected on to a DIOL column and eluted with an AcOEt/Et.sub.2O gradient (from 0 to 50%). The fractions of interest (furylated extension units) are combined and evaporated under vacuum. 54 mg of the furylated flavan monomer below are obtained:

##STR00024##

[0199] The NMR data obtained for this compound, indicated in table 5 below, confirm the structure above.

TABLE-US-00005 TABLE 5 NMR characterization of the furylated derivative obtained by means of a depolymerization method according to the invention Attribution .sup.13C (ppm) Type .sup.1H (ppm) J (Hz) 1 74.4 CH 4.57 sl 2 68.4 CH 3.94 m 3 39.1 CH 4.07 m 4 98.2 Q — — 5 155.9 Q — — 6 94.0 CH 5.78 d-2.1 Hz 7-9-10 157.3-157.1-157.0 Q — — 8 95.4 CH 5.92 d-2.1 Hz 11 106.6 CH 5.80 d-2.9 Hz 12 110.3 CH 6.34 dd-1.7/2.9 Hz 13 141.4 CH 7.55 d-1.7 Hz 14 130.1 Q — — 15 117.7 CH 6.51 dd-1.7/8.1 Hz 16 114.9 CH 6.66 d-8.1 Hz 17-18 144.7-144.6 Q — — 19 114.7 CH 6.81 d-1.7 Hz 20-21 OH 8.72-8.82 s 22 OH 9.04 s 23 OH 9.15 s 24 OH 5.08 d-4.7 Hz

[0200] The positions of the aromatic groups are moreover confirmed by .sup.13C-.sup.1H HMBC heteronuclear two-dimensional NMR.

EXAMPLE 8

Preparative Depolymerization of White Seed Tannins with Sylvan

[0201] A depolymerization method in accordance with the invention is carried out in the following way.

[0202] In a round-bottomed flask, the tannin extract (8.0 g) is dissolved in MeOH (300 ml), then sylvan (100 ml), and then gently fuming HCl (3.33 ml), are added with stirring. The mixture is brought to 30° C. for 60 min. 400 ml of an aqueous solution of Na.sub.2CO.sub.3 (5.3 g.l.sup.—1) are added and then an extraction is carried out with AcOEt (3×400 ml). The solution is evaporated so as to give a pasty brownish solid (5.7 g), which is taken up with Et.sub.2O (200 ml), triturated and sonicated, and then washed with brine (300 ml). These operations are repeated twice, and the resulting solutions are combined, and dried with Na.sub.2SO.sub.4. The solution is evaporated so as to obtain a bullate beige solid (2.40 g), consisting of a mixture of terminal units and of sylvanylated extension units.

[0203] The product thus obtained is purified by flash chromatography. The mixture (300 mg) is dissolved in Et.sub.2O (5 ml), and is then injected on to a DIOL column and eluted with an AcOEt/Et.sub.2O gradient (from 0 to 50%). The fractions of interest (sylvanylated extension units) are combined and evaporated under vacuum. 99 mg of the sylvanylated flavan monomer below are obtained:

##STR00025##

[0204] The NMR data obtained for this compound, indicated in table 6 below, confirm the structure above.

TABLE-US-00006 TABLE 6 NMR characterization of the sylvanylated derivative obtained by means of a depolymerization method according to the invention Attri- bution .sup.13C (ppm) Type .sup.1H (ppm) J (Hz)  1 74.3 CH 4.61 sl  2 68.4 CH 3.92 m  3 39.1 CH 4.03 m  4 98.2 Q — —  6 94.0 CH 5.77 d-2.5 Hz 5-7-9-10 157.3-157.0-156.0-155.3 Q — —  8 95.3 CH 5.92 d-2.3 Hz 11 106.3 CH 5.92 d-2.5 Hz 12 107.3 CH 5.62 d-2.5 Hz 13 149.8 Q — — 14 130.1 Q — — 15 117.8 CH 6.52 dd-1.9/7.9 Hz 16 114.9 CH 6.67 d-8.1 Hz 17-18 144.7-144.6 Q — — 19 114.7 CH 6.81 d-2.0 Hz 20-21 — OH 8.72-8.82 s 22 — OH 9.03 s 23 — OH 9.13 s 24 — OH 5.05 d-5.0 Hz .sup. 14′ 15.2 CH 2.24 sl

[0205] The positions of the aromatic groups are moreover confirmed by .sup.13C-.sup.1H HMBC heteronuclear two-dimensional NMR.

EXAMPLE 9

Stability Under Basic Conditions of Derivatives Obtained by Thiolysis or by Sylvanolysis

[0206] A buffer solution at pH 9 is prepared by adding 50 ml of a solution containing 0.2 mol/l of KCl and 0.2 mol/l of H.sub.3BO.sub.3 to 21.4 ml of a solution of NaOH at 0.2 mol/l.

[0207] Thiolysis

[0208] An aqueous solution containing 2 g/l of a crude product resulting from the depolymerization of a white seed tannin extract by thiolysis according to the protocol of Roumeas et al., 2013 is prepared. This solution contains the catechin (Cat) and epicatechin (Ec) terminal units, and also the sulfur-containing extension units derived from catechin (Cat-M) and from epicatechin (Ec-M). The UPLC chromatogram at 280 nm of this solution is shown in FIG. 6(a).

[0209] 500 μl of this solution are transferred into a vial to which are added 500 μl of the buffer solution at pH 9. The vial is flushed with argon, sealed and left in the dark for 19 h. The UPLC chromatogram at 280 nm, of the resulting solution, is shown in FIG. 6(b). The disappearance of the peaks corresponding to the sulfur-containing extension units is observed therein. An analysis by UPLC-MS(+) shows that these sulfur-containing extension units have been more than 95% degraded in 19 h.

[0210] Sylvanolysis

[0211] In a first experiment, an aqueous solution containing 2 g/l of a crude product resulting from the depolymerization of a white seed tannin extract with the sylvan according to the protocol described in example 8 is prepared. This solution contains the catechin (Cat), epicatechin (Ec) and epicatechin gallate (EcG) terminal units, and also the sylvanylated extension units (2-methylfurylated) derived from catechin (Cat-S), from epicatechin (Ec-S) and from epicatechin gallate (EcG-S). The UPLC chromatogram at 280 nm of this solution is shown in FIG. 7(a).

[0212] 500 μl of this solution are transferred into a vial to which are added 500 μl of the buffer solution. The vial is flushed with argon, sealed and left in the dark for 19 h. The UPLC chromatogram at 280 nm, of the resulting solution, is shown in FIG. 7(b). It is observed therein that the peaks corresponding to the sylvanylated extension units are always present. An analysis by UPLC-MS(+) shows that there was no degradation of the product after 19 h.

[0213] In a second experiment, the same product resulting from depolymerization of white seed tannins by sylvanolysis is dissolved at a concentration of 1 g/l in a solution of acetonitrile containing triethylamine at two concentrations, 0.02 or 1.8 mM (at these concentrations, the triethylamine in aqueous phase would form solutions at pH 10 and at pH 11, respectively). The two solutions are left under argon for 4 days in the dark in sealed vials. The analysis thereof by UPLC-MS(+) shows no degradation after 4 days.

EXAMPLE 10

Determination of the “Nucleophile/Depolymerizable Condensed Tannins” Molar Ratios for Furan and Sylvan

[0214] A test carried out using a model tannin, the dimer B2, and either sylvan or furan as nucleophile, was developed in order to determine the minimum “nucleophile/depolymerizable condensed tannins” molar ratios to be used in the condensed tannin depolymerization reactions. In order to obtain a discriminating result, the test is based on the measurement of the rate of conversion with respect to units of derivatives of interest (furylated derivative or sylvanylated derivative) over a short reaction time, that is to say 10 min. Consequently, the rat measured in this test on initial reaction rates does not correspond to the degree of conversion obtained at the end of the reaction owing to the reaction kinetics. However, the comparison of the results of the test on the dimer B2 with those of the experiments which were carried out under real conditions on the industrial extract of white seed tannins for a maximum period of 8 h while retaining the same conditions of temperature, acidity and “nucleophile/tannins expressed in monomer equivalent” molar ratio outside this test and the results of which are presented in tables 2 and 4 of this document, makes it possible to establish a correlation between the degree of conversion in the test and the yields of production of the compounds of interest obtained under real conditions. Thus, a threshold lying at around 2.5% for the degree of conversion in the context of the test makes it possible to obtain, under the real production conditions, yields of compounds of interest of about 40%. The curve which is determined by the test presented below then makes it possible to determine the minimum values of the “nucleophile/dimer B2 in monomer equivalent” molar ratios which correspond to the minimum “nucleophile/depolymerizable tannins in monomer equivalent” molar ratios that will have to be used in the depolymerization reactions under real conditions.

[0215] Described below is the procedure used to determine the “nucleophile/depolymerizable condensed tannins” molar ratio using a given tannin extract, taking furan or sylvan as nucleophile.

[0216] For an extract of given tannins, the molar amount of depolymerizable condensed tannins is determined by analytical thiolysis reaction, carried out in particular as described in the publication by Roumeas et al., 2013, and quantification of the amount of tannins thus depolymerized, by UPLC-MS, as indicated above.

[0217] The corresponding molar amount of nucleophile of general formula (III) is determined in the following way, for furan and sylvan.

[0218] A stock solution of dimer B2 is prepared at 100, 200 or 400 mg.l.sup.−1 in MeOH (reference concentration: 400 mg.l.sup.−1, that is to say 0.7 mM). The depolymerization solutions contain 200 mM of HCl and various concentrations of nucleophile of between 0.7 and 70 mM for sylvan, and 140 and 7000 mM for furan.

[0219] For each experiment, a mixture of 500 μl of stock solution of dimer B2 and 500 μl of depolymerization solution is prepared in a stoppered tube and immediately brought to 30° C. for 10 min. The samples are then directly analyzed by UPLC, according to the protocol described above, with neither treatment nor dilution beforehand. The quantification of the extension units U.sub.ext (in derivative form) and of terminal units U.sub.term released during the reaction makes it possible to calculate, for each test, the U.sub.ext/(U.sub.ext+U.sub.term) ratio.

[0220] The results obtained, for each of furan and sylvan, as a function of the various “nucleophile/dimer B2 in monomer equivalents” molar ratios, are indicated in table 7 below.

TABLE-US-00007 TABLE 7 U.sub.ext/(U.sub.ext + U.sub.term) molar ratios obtained by depolymerization of dimer B2 with furan and sylvan in accordance with the present invention “Nucleophile/dimer Sylvan U.sub.ext/(U.sub.ext + U.sub.term) Furan U.sub.ext/(U.sub.ext + U.sub.term) B2” molar ratio molar ratio molar ratio 0.5 0.033* — 2.5 0.089* — 0.5 0.014** — 0.5 0.020* — 5 0.167* — 5 0.080*** — 5 0.140* — 10 0.170* — 50 0.420*** — 50 0.320* — 100 — 0.035* 250 — 0.070* 500 0.470** 0.135* 500 0.500* — 500 0.500*** — 3750 — 0.430*** 5000 — 0.420* with: *experiments carried out in the presence of 0.35 mM of dimer B2 **experiments carried out in the presence of 0.087 mM of dimer B2 ***experiments carried out in the presence of 0.17 mM of dimer B2

[0221] On the basis of these results, the curves representing the U.sub.ext/(U.sub.ext+U.sub.term) molar ratio as a function of the “nucleophile/dimer B2” molar ratios are plotted. The curves obtained, for furan and sylvan, are shown in FIG. 8.

[0222] The results obtained demonstrate the competition reactions which take place between the nucleophiles and the flavonoid compounds present in the medium. They thus allow a comparison of the power of the nucleophiles with one another. Thus, the figure clearly shows that sylvan is a better nucleophile than furan, in so far as similar degrees of conversion with respect to extension units (U.sub.ext/(U.sub.ext+U.sub.term) molar ratio) are achieved for “nucleophile/dimer B2” molar ratios that are much lower in the case of sylvan.

[0223] For example, using as a basis the smoothing curves obtained from the experimental points, shown in FIG. 8, the “nucleophile/dimer B2” molar ratio required to achieve, under the conditions of the test, a degree of conversion of 2.5% with respect to extension units, relative to the total of the extension units and of the terminal units, can be established at 0.79 for sylvan, and at 63 for furan. The “nucleophile/dimer B2” molar ratio required to achieve, under the conditions of the test, a degree of conversion of 5% with respect to extension units, relative to the total of the extension units and of the terminal units, can be established at 1.67 for sylvan, and at 133 for furan.

[0224] The same experiment can be carried out, under similar conditions, for any other nucleophile of general formula (III) according to the invention.

EXAMPLE 11

Depolymerization of Tannins Directly from Douglas Pine Bark

[0225] Comparative Example—Thiolysis

[0226] A thiolysis reaction, according to the protocol described in the publication by Roumeas et al., 2013, is carried out in the following way.

[0227] In a 100 ml round-bottomed flask, the Douglas pine bark (800 mg) is suspended in 40 ml of mercaptolysis solution (2.0 ml of 2-mercaptoethanol and 333 μl of fuming HCl per 40 ml of MeOH qs), and left to stir for 2 h at 40° C. The results obtained by UPLC-MS show a maximum content of extension unit of 129 μmol.g.sup.−1 of initial bark.

[0228] Depolymerization Process with Sylvan in Accordance with the Invention

[0229] In a round-bottomed flask, the Douglas pine bark milled to 6 mm (10.0 g) is suspended in MeOH (75 ml), then sylvan (25 ml) and then fuming HCl (833 μl) are added with stirring. The mixture is brought to 30° C., and the reaction is monitored by UPLC-MS. After 2 h 30, the sylvanylated derivative yield reaches a maximum of 119 μmol.g.sup.−1 of bark, that is to say 92% of the value obtained by means of the comparative thiolysis reaction.

[0230] The targeted sylvanylated derivatives are thus obtained with a high yield, directly from the bark, without prior tannin extraction.

[0231] Preparative Extraction

[0232] The reaction mixture obtained by means of the sylvanolysis reaction is filtered through a Büchner funnel in order to remove the bark, and 100 ml of an aqueous solution of NaHCO.sub.3 (8.4 g.l.sup.−1) are added. The solution obtained is evaporated under vacuum in order to remove the methanol and the sylvan, and the aqueous suspension is extracted with AcOEt (3×100 ml). The organic phase is dried over Na.sub.2SO.sub.4 and evaporated under vacuum to give an oily orangey solid (1.25 g). This product is triturated from petroleum ether (3×50 ml) in order to remove the lipids and the terpenes, and then taken up in Et.sub.2O (140 ml). The suspension is washed with brine (2×50 ml), then dried over Na.sub.2SO.sub.4 and evaporated under vacuum to give an orangey solid (920 mg) comprising 291 mg of sylvanylated derivatives.

LITERATURE REFERENCES

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